Gene involved in CADASIL, method of diagnosis and therapeutic application

ABSTRACT

The invention concerns the Notch3 gene and the corresponding protein, which are involved in CADASIL. The invention concerns, in particular, methods for demonstrating mutations in this gene, which are linked to the risk of developing CADASIL. The invention also concerns models and products for treating CADASIL and related diseases.

This is a division of application Ser. No. 09/230,652, filed Jul. 31, 1997, as PCT/FR97/01433, and which entered the U.S. National Stage on Jan. 29, 1999. U.S. application Ser. No. 09/230,652 is hereby incorporated in its entirety herein by reference.

The present invention relates to the demonstration of the involvement of the Notch3 protein in CADASIL thus allowing in particular a diagnosis of a predisposition to certain neurological disorders, in particular CADASIL, and models which make it possible to test the therapies possible for this type of pathology.

CADASIL or “Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy” has recently been identified as a cause of cerebral attacks and of dementia whose main characteristics include recidivous subcortical infarcts, migraines and a vascular dementia, in association with MRI images objectivizing diffuse abnormalities of the cerebral white substance.

An anatomicopathological examination shows multiple small deep cerebral infarcts, a leukoencephalopathy and a nonatherosclerotic and nonamyloid angiopathy involving essentially the small cerebral arteries.

As its name indicates, CADASIL is a hereditary disease with a dominant autosomal character. For more information, there may be found in particular a study of the clinical spectrum of CADASIL in H. Chabriat et al., The Lancet, Vol. 346, 7 Oct. 1995.

This highly incapacitating and very often lethal disease has probably remained so far largely undiagnosed as such; the study of about one hundred families since 1993 shows that erroneous diagnoses were most often given to the patient (multiple sclerosis, Alzheimer's disease and the like). Current studies would tend to demonstrate that it is a condition which is much more widespread than what was thought during its discovery.

The research studies currently pursued have the objective of identifying diagnostic tools for the disease and, by virtue, in particular of the models and the possibilities offered by genetic engineering, of developing a possible substitute therapy.

The gene involved in CADASIL has been localized on chromosome 19 and a finer localization is in particular mentioned in two patent applications with the same inventors.

It has now been possible to identify the gene involved in CADASIL which is the Notch3 gene.

The demonstration of the involvement of Notch3 in CADASIL has been possible given the previous limits which had been mentioned especially in the patent applications in question, the first interval (size 14 cM) was D19S221-D19S215 (first patent application), and then the second interval (size 2 cM) was D19S226-D19S199 (second patent application). The region of interest was cloned into a BAC and YAC contig (continuous nucleotide sequence) and its size was estimated at 800 kb. Analysis of this region with the aid of restriction enzymes showed a very high density of NotI, EagI and SacII sites which suggested the presence of numerous genes. Among the numerous transcripts identified by cDNA selection, one transcript showed a very strong homology with a sequence situated at the coding 5′ end of the mouse gene Notch3. Since other analytical factors seemed to corroborate this presence of the Notch3 gene in this situation, the latter was considered to be a good candidate gene by its position in the interval of interest.

The comparative studies carried out on known CADASIL families in comparison with healthy subjects have made it possible to identify mutations on this Notch3 gene in a large number of CADASIL subjects whereas such mutations were not observed on the healthy subjects analyzed. Since, finally, it has been possible to demonstrate the cosegregation of these mutations with the disease phenotype within effected families, the involvement of the Notch3 gene in CADASIL became incontestable.

All the point mutations observed lead to the creation or to the disappearance of a cysteine in one of the EGF domains of this protein. These mutations are clustered for a large part of them into the first six EGFs. The clustering of the mutations is certainly important in diagnostic terms especially for the “sequential” search for these mutations.

Moreover, all these mutations lead to the presence of an odd number of cysteines in one of the EGFs (either seven, or five cysteines) instead of the six cysteines normally present. These mutations could thus result in the formation of either intra- or intermolecular (and in this case in the formation of homo- or heterodimers) aberrant disulfide bridges.

The role of a normal or abnormal dimerization in the functioning of receptors, in particular their activation, is well known.

The Notch genes have been known for a very longs time, especially in drosophila and their equivalent is known in vertebrates, in particular in mice. Its English name “notch” comes from the fact that some mutations of this gene produce a notch in the wings of flies. The article by Spyros Artavanis-Tskanas et al., Science 268, 225 (1995) as well as the references which it contains indicate that the Notch proteins are essentially involved, especially in drosophila, in the specification of the cellular destiny during development, and although the protein is always expressed in adult organisms, its functions in the latter remain unknown. More precisely, it appears that the product of the Notch3 gene, hereinafter “Notch3 protein”, is a cell receptor which controls a cascade of cellular events and whose mutation necessarily leads to greater or lesser disruptions in this cascade which may lead to many other neurological, especially cerebrovascular, disorders.

It should be recalled that, while in the text which follows there is interest more particularly in neurological disorders, in particular cerebrovascular-type disorders and most particularly CADASIL, it is probable, given the function of the cell receptor for the product of the Notch3 gene, that impairment of this receptor can lead to a disorganization of its interaction with various ligands but also with the various partners involved in the transduction cascade. Account should be taken, in addition, of the fact that the Notch3 protein might have other functions which have not yet been demonstrated. Under these conditions, it is highly probable that conditions exhibiting similarities with CADASIL may also be involved in the case of a mutation in the Notch3 gene.

Among the relevant diseases, there may be mentioned the sporadic forms of CADASIL, that is to say which occur without a family history but following a neomutation. Notch3 might moreover be involved in other conditions which may be classified into different groups:

Migraine and Hemiplegic Migraine

It was shown that at least one of the genes involved in familial hemiplegic migraine (FHM), the dominant autosomal form of migraine with aura, was located in the same region of chromosome 19—as the CADASIL gene. It should be noted that more than 30% of patients suffering from CADASIL, a condition characterized by the repeated onset of cerebrovascular accidents and of a vascular dementia, suffered from migraine with aura. However, the latter is observed in only about 5% of the population; it is this observation which led to testing the involvement of the CADASIL gene in the mechanisms of this condition. The involvement of this gene in a form of migraine with or without aura was of considerable diagnostic and therapeutic interest because of the frequency of migraine with aura and of migraine without aura in the general population.

Other Vascular (Cerebral Infarct) and/or Dementia Pathologies of Unknown Etiology

This group corresponds to a very large number of patients in neurology, psychiatric and internal medicine departments and it is everything to do reasonable to think that Notch3 or a partner in this signaling pathway may be involved in these conditions for the reasons stated above.

Familial Paroxytic Ataxia

The situation is the same as for FHM. A genes responsible for this condition has been located in the same region of chromosome 19 and Notch3 could be implicated in this condition.

Moreover, the mutations of this gene are responsible for developmental abnormalities which are well known in other species as well as for neoplastic-type pathologies. Malformative and/or neoplastic syndromes in which there may be demonstrated a rearrangement of the region which contains this gene might be major candidates for the search for an involvement of this gene in their physiopathology.

These disorders may be grouped under the name of “disorders linked to the Notch3 receptor”.

In some cases, this may involve disorders having a multigenic origin but in which the modifications of Notch3 might contribute to the onset of the pathology or to its worsening.

The present invention relates, first of all, to an isolated nucleotide sequence, characterized in that it is chosen from:

-   a) the sequences encoding the human Notch3 protein and its allelic     variants, -   b) the sequences encoding a fragment of these proteins and having at     least 10 bases, -   c) the human Notch3 genomic sequences and its alleles, -   d) the sequences exhibiting at least 80%, and preferably at least     90%, homology with the sequences (a) and (c), -   e) the fragments of the sequences (c) or (d) having at least 10     bases, -   f) the sequences which hybridize with a sequence of (a) to (e).

It should be understood that the present invention does not relate to the genomic nucleotide sequences in their natural chromosomal environment, that is to say in the natural state; they are sequences which have been isolated, that is to say that they were collected directly or indirectly, for example by copying (cDNA), their environment having been at least partially modified.

Thus, this may also involve both cDNA and genomic DNA which is partially modified or carried by sequences which are at least partially different from the sequences carrying them naturally.

These sequences may also be described as being “normatural”.

“Nucleic sequence” is understood to mean a natural isolated, or synthetic, fragment of DNA and/or RNA designating a precise linkage of nucleotides, modified or. otherwise, making it possible to define a fragment, a segment or a region of a nucleic acid.

“Allelic variant” of the protein is understood to mean all the mutated proteins and the polymorphisms which may exist in a human being, which are obtained in particular by truncation, substitution, deletion or addition of amino acid residues, as well as the artificial variants.

According to the invention, the nucleic sequence fragments may in particular encode domains of the protein or may be used as probe or as primer in methods of detection, identification or amplification. These fragments have a minimum size of 10 bases and fragments of 20 bases, preferably 30 bases, will be preferred.

According to the invention, the homology is solely of the statistical type; it means that the sequences exhibit at least 80%, and preferably 90%, of nucleotides in common.

The hybridization conditions should make it possible, according to the invention, to ensure at least 95% homology.

More particularly, the present invention relates to a nucleotide sequence chosen from:

-   a) the sequences encoding a polypeptide comprising the amino acids     according to the sequence in FIG. 1, -   b) the nucleic sequences corresponding to FIG. 1, -   c) a fragment of a sequence according to (a) or (b) containing at     least 10 bases, and -   d) a sequence which contains, relative to the sequences (a), (b) or     (c), at most 20 partial mutations.

FIG. 1 represents the sequences of Notch3 as were sequenced on a normal genome.

The sequences are identified by references which make it possible to position them relative to each other using FIG. 3.

As regards the special remarks on (a), (b), (c) and (d), the previous remarks apply.

The invention also relates to the fragments of these sequences, in particular sequences encoding polypeptides which have retained all or part of the activity of the Notch3 protein.

Among the particularly advantageous sequences, there may be mentioned those encoding domains or combinations of domains of the Notch3 protein, that is to say the sequences:

-   -   “EGF” repeats     -   “Notch/lin12” repeats     -   “cdc10/SW16” repeats or the transmembrane sequence.

Among the advantageous sequences are in particular the sequence encoded by the second transcript which will be described in the text which follows, the said transcript having an estimated size of between 1.3 and 2.4 kb.

These sequences may be identified with reference in particular to FIG. 2 which schematically represents the organization of Notch3.

These partial sequences can be used for numerous applications, as described below, especially for preparing Notch-type or different types of protein constructs, but also for preparing, for example, truncated Notch-like proteins which will serve as lure for the Notch3 ligand or as agonist for the protein.

It is also possible to envisage using these protein sequences for their intrinsic effects; thus, the EGF domains are present in other proteins, especially other receptors; reference may be made for example to Iain D. Campbell, Current Biology, 3: 385–392 (1993) for other applications of the EGF sequences in question.

While the sequences described are in general normal sequences, the invention also relates to the mutated sequences insofar as they contain at least one point mutation and preferably less than 20 mutations in total.

Preferably, the present invention relates to the nucleotide sequences in which the point mutations are not silent, that is to say that they lead to a modification of the amino acid encoded relative to the normal sequence. Still more preferably, these mutations affect amino acids which structure the Notch3 protein or the corresponding fragments thereof, that is to say in particular the mutations which suppress the cysteines or, on the contrary, which make them appear, but also the mutations which change the character of the protein, either from the charge point of view, or from the hydrophobicity point of view.

The present invention also relates to the mutations which may occur in the promoter and/or regulatory sequences of the human Notch3 gene, which may have effects on the expression of the protein.

Examples of such mutations will be described in the text which follows.

In general, the present invention relates to both the normal Notch3 protein and the mutated Notch3 proteins, as well as to their fragments and to the corresponding DNA and RNA sequences, that is to say the alleles.

It should be noted that the Northern blot study of the expression of this gene in human tissues reveals two transcripts. One having a size estimated at 7.5–9.5 kb is present in all the tissues tested; the other, whose size is between 1.3 and 2.4 kb, is detected only in some parts of the central nervous system. The present invention relates to these two transcripts.

Among the nucleotide fragments, there may be mentioned the intron genomic sequences of the Notch3 gene and more particularly the joining sequences between the introns and the exons, especially as are represented in Table A; and finally, the present invention relates to all the primers which may be deduced from the preceding nucleotide sequences and which may make it possible to detect them using an amplification method such as the PCR method, especially those presented in Table B.

The present invention also relates to the nucleotide sequences which may contain nonnatural nucleotides, especially sulfur-containing nucleotides for example or having an α or β structure.

Finally, the present invention of course relates to both the DNA and RNA sequences, as well as the corresponding double-stranded DNAs.

As will be described below for some applications, it may be necessary to provide for mixed constructs, protein/DNA/chemical compound, especially the use of intercalating agents for example; it should be understood that such compounds are covered by the patent as containing a sequence according to the invention.

The present invention also relates to the polypeptide or peptide proteins corresponding to the abovementioned sequences, in a nonnatural form, that is to say that they are not taken in their natural environment but obtained by purification from natural sources or obtained by genetic recombination, as will be described below.

The invention also relates to the same polypeptides or proteins obtained by chemical synthesis and capable of containing nonnatural amino acids.

The present invention relates to the recombinant proteins thus obtained both in a glycosylated and nonglycosylated form and capable of having or otherwise the natural tertiary structure.

In particular, the present invention relates to the Notch3 fragments which exhibit an activity similar to the total receptor, especially the soluble part(s) of said receptor corresponding in particular to their extracellular domain. These may be used especially as a lure in a therapy, as will be described below.

The present invention also relates to the cloning and expression vectors containing a nucleotide sequence as described above.

These cloning and expression vectors may contain elements ensuring the expression of the sequence in a host cell, especially promoter sequences and regulatory sequences which are efficient in said cell (see reference below).

The vector in question may be autonomously replicating or intended to ensure the integration of the sequence into the chromosomes of the host cell.

In the case of autonomously replicating systems, depending on the prokaryotic or eukaryotic host cell, plasmid-type systems or viral systems will preferably be used, it being possible for the viral vectors to be especially adenoviruses, poxviruses or herpesviruses. Persons skilled in the art know the technologies which can be used for each of these viruses (see reference below).

When the integration of the sequence into the chromosomes of the host cell is desired, it will be necessary to provide for, on either side of the nucleotide sequence to be integrated, one or more sequences obtained from the host cell in order to bring about the recombination. These are also methods which are widely described in the prior art. It will be possible, for example, to use plasmid or viral type systems; such viruses will be, for example, retroviruses or AAVs (Adeno-Associated Viruses).

The invention also relates to the prokaryotic or eukaryotic cells transformed by a vector as described above, and this being in order to bring about the expression of a natural or mutated Notch3 protein or, for example, of one of its subunits.

As indicated above, the present invention also relates to the proteins, peptides or polypeptides obtained by culturing the cells thus transformed and recovering the protein expressed, it being possible for said recovery to be carried out intracellularly or extracellularly from the culture medium when the vector has been designed to bring about the excretion of the protein via for example a “leader” sequence, the protein being in a pre-protein or prepro-protein form. The constructs allowing the secretion of the proteins are known both for prokaryotic systems and eukaryotic systems.

Among the cells which can be used for the production of these proteins, there may of course be mentioned bacterial cells, but also yeast cells, as well as animal cells, in particular mammalian cell cultures, but also insect cells in which methods using baculoviruses for example may be used (see reference below).

The cells thus obtained can make it possible to prepare natural or mutated Notch3 proteins, but also fragments of these proteins, especially polypeptides which may correspond to the different domains in question.

However, the cells transformed as described above may also be used as a model to study the interactions between the Notch gene and its various ligands as well as its influence on the products downstream of the receptor, but in particular they may be used in an application for the selection of products interacting with the natural or mutated Notch3 receptor, as an agonist or an antagonist of this receptor.

This type of cellular model may be produced using genetic engineering techniques. It involves, depending on the type of cells which it is desired to use, cloning the gene in question in its normal form or in its mutated form into an expression vector, whether it is an. autonomously replicating vector or an integration vector, said vector containing all the elements allowing the expression of the gene in the cell in question, or the latter having all the elements allowing the expression of the sequence in question.

There are thus obtained eukaryotic or prokaryotic cells expressing the Notch3 protein(s) which, given its characteristics, will be situated like a transmembrane protein whose fine structure will be described in the text which follows, it being possible for said cells to then constitute models which make it possible to test at the same time the interactions Of various ligands with the product of the Notch3 protein or to test synthetic chemical products capable of interacting with the product of the Notch3 gene, and this by adding them to the culture medium for said cells.

It should in particular be noted that the products in question may also be products with either antagonist or agonist activity.

The use of cellular models to test pharmaceutical products is well known; here again, there is no need to present this type of model in detail.

Another potential application of the characterization of this gene is the possibility of identifying potential ligands for this protein, either because they have a conserved sequence with human Notch3, or because they interact with Notch3 (affinity methods) or partners for this signaling pathway.

These models may be of the in vitro type, for example cultures of human cells, either in a normal culture, or possibly in the form of an isolated organ, such as for example certain types of vessels which may be transformed in order to cause them to express the desired phenotypes.

The present invention also relates to the organisms, such as animals, in particular mice, expressing the phenotype corresponding to the normal or mutated Notch3 of human origin. Here again, these animals may be used as model animals to test the efficacy of certain pharmaceutical products.

The present invention also relates to the products obtained using the preceding cellular models.

There will thus be obtained, depending on the type of interaction determined, therapeutic compositions characterized in that they contain, as active ingredient, a compound with a pro-Notch3 activity; this may be in particular all or part of a polypeptide as were described above or a vector expressing these same polypeptides, or else chemical or biological compounds having a pro-Notch3 activity, a Notch3-like activity or inducing the production of natural Notch3.

It will also be possible to demonstrate therapeutic compositions in which the active ingredient will have an anti-Notch3 action.

This may involve, here again, modified proteins described above which may play the role of a lure, or anti-Notch3 antibodies, in particular when these antibodies recognize the mutated receptors and will, under these conditions, be able to block the activity of the normal receptor.

This may also involve chemical products having an anti-Notch3 activity, or Notch3 antagonists.

In some cases, the use of some of the Notch3 domains may allow a therapeutic approach blocking the activity of the Notch3 receptor when the latter is mutated using soluble receptors which will serve as lure to the natural ligands; in other cases, it will be possible, by expressing the entire receptor, to provide a replacement therapy using either directly the proteins or fragments thereof, or directly expressing the protein, especially via gene therapy and using the vectors which were described above.

In the context of gene therapy, it is also possible to provide for the use of the sequences of the genes or cDNAs described above as “naked”; this technique was in particular developed by the company Vical; it has shown that it was possible, under these conditions, to express the protein in certain tissues without requiring the use of the support for a viral vector in particular.

Still in the context of gene therapy, it is also possible to provide for the use of cells transformed ex vivo, which cells may then be reimplanted either as such or in systems of the organoid type, as is also known in the state of the art. It is also possible to envisage the use of an agent facilitating targeting of the determined cell type, penetration into the cells or transport to the nucleus.

Among the numerous pharmaceutical compounds which can be used, there should be mentioned more particularly, in addition to the ligands for the Notch3 product, the sense or anti-sense sequences interacting with the normal or mutated Notch3 gene, or interacting on the regulation or expression of these genes, it being also possible for said products to interact downstream of the expression products induced by the Notch3 receptors. The soluble sequences corresponding to Notch3 should furthermore be cited.

There should also be mentioned the monoclonal antibodies blocking the Notch3 receptors, in particular the mutated Notch3 receptors, and/or blocking the corresponding ligands and/or the products induced by said receptors which may therefore have pro or anti activities.

It should be recalled that the monoclonal antibodies directed against the Notch3 receptor may, depending on the epitope recognized, have a pro or anti-Notch3 activity which makes them useable in therapeutic compositions.

Finally, the present invention relates, as was said above, more particularly to the methods of diagnosing a predisposition to neurological conditions, especially of the CADASIL type, or of diseases linked to the Notch3 receptor in a patient, characterized in that the presence of a mutation in Notch3 is determined using a biological sample from said patient by analysis of all or part of a nucleic sequence corresponding to said gene, the presence of at least one such mutation being indicative of a predisposition of said patient to neurological conditions or diseases linked to the Notch3 receptor.

Other diagnostic methods can make it possible to characterize, by means of antibodies, the deposit expected in the basal membrane of the vascular smooth muscle cells, a deposit which might consist of the Notch3 protein itself or one of its cleavage products.

Among the desired mutations, there may be mentioned more particularly the mutations referenced in Table C and FIG. 3.

The nucleic acid sequences may be either genomic DNA, a cDNA or an mRNA.

As was said above, among the neurological disorders which may be demonstrated, there is understood more particularly disorders of the cerebrovascular type and especially CADASIL, but the list of certain disorders which might be linked to an abnormality in the Notch3 receptor has been previously given; among these conditions, there may be mentioned most particularly the potential involvement of Notch3 in migraines with or without aura and dementias of currently unknown etiology.

The diagnostic tools based on the present invention may allow a positive and differential diagnosis in a patient taken in isolation or alternatively a presymptomatic diagnosis in an at-risk subject (family history for example), it is also possible to envisage an antenatal diagnosis.

In addition, the detection of a specific mutation may allow an evolutive diagnosis.

The methods which make it possible to demonstrate the mutation in a gene relative to the natural gene are of course very numerous; they may be carried out by studying the genomic DNA, the cDNA and/or the protein. They can be essentially divided into two large categories, the first type of method is that in which the presence of a mutation is detected by comparing the mutated sequence with the corresponding nonmutated natural sequence, and the second type in which the presence of the mutation is detected indirectly, for example, by detecting the mismatches due to the presence of the mutation.

In both cases, the methods in which all or part of the sequence corresponding to Notch3 is amplified prior to the detection of the mutation will be preferred in general; these amplification methods may be carried out by the so-called PCR (see reference below) or PCR-like methods. PCR-like will be understood to designate all the methods using direct or indirect reproductions of the nucleic acid sequences, or in which the labeling systems have been amplified; these techniques are well known, in general they relate to the amplification of DNA by polymerase; when the original samples is an RNA, it is advisable to carry out a reverse transcription beforehand; a great number of methods allowing this amplification currently exists, for example the so-called NASBA and TMA methods which are well known to persons skilled in the art.

Table B gives the sequences of the PCR primers which make it possible to amplify the exons as well as the temperatures for the PCR reactions.

A general methodology for amplification of the sequences will be described in the examples.

Test for Point Mutations

In addition to the direct sequencing of the mutation, various methods may be used. The techniques will be briefly cited:

-   1) test for “Single Strand Conformation Polymorphisms” (SSCP) (see     reference below) or denaturing gradient gel electrophoresis (DGGE). -   2) the methods based on a cleavage of the mismatched regions     (enzymatic cleavage by S1 nuclease, chemical cleavage by various     compounds such as piperidine or osmium tetroxide, and the like. -   3) heteroduplex detection by electrophoresis, -   4) methods based on the use in hybrication of allele-specific     oligonucleotide (ASO) probes.

Other well known methods based on hybrication techniques can be used.

Test for Deletion, Inversion or Duplication Type Rearrangements

Other well known methods based on the techniques of hybridization with the aid of genomic probes, of cDNA probes, of oligonucleotide probes, of riboprobes, of so-called capture probes or of so-called detection probes, may be used for the test for this type of rearrangement.

Another diagnostic approach which can be used when DNA from several subjects of the same family is available is based on the method of linkage analysis which makes it possible to calculate the risk which a subject belonging to a linked family has of being a carrier or otherwise of the diseased gene. This analysis may be carried out with polymorphic markers situated in the immediate vicinity of the gene, or intragenic polymorphic markers.

It is important to recall that in the CADASIL families, the existence of mutations in the Notch3 gene corresponds to mutations which change amino acids which are essential for the function of the protein for which it encodes.

Moreover, in the examples, the situations of the mutations currently detected are indicated, but it is possible that other mutations exist in the Notch3 gene which have not yet been detected but which should lead to the same types of risks from the pathological point of view.

In any case, the mutated Notch3 proteins may exhibit an antigenicity which is different from that of the natural protein.

It is therefore possible to carry out a diagnosis or a prognosis of a susceptibility to neurological, in particular cerebrovascular, disorders of the CADASIL type and disorders linked to the Notch3 receptor, by detecting the product of the mutated gene for Notch3; this type of detection can be carried out, for example, with the aid of monoclonal or polyclonal antibodies. Under these conditions, it is possible to detect and assay the abnormal product of the Notch3 gene by well known methods, RIA or ELISA for example; these technologies being known, they will not be further developed beforehand in the text which follows. Antibodies directed against the normal protein could also be used if the deposit present in the arteries of the skin corresponded to the Notch3 protein or to one of its cleavage products.

The present invention also relates to the labeled monoclonal or polyclonal antibodies corresponding to all or part of the mutated proteins so as to serve as imaging agent in vivo or ex vivo on biological samples.

Thus, it appears that the granular masses present in the basals of the vascular smooth muscle cells are due to an accumulation of the abnormal protein and the test for this protein with the aid of antibodies, either in biopsies or in vivo, is of a diagnostic interest.

Methods Based on the Detection of the Product of the Gene

The mutations of the Notch3 gene may be responsible for various modification of the product of this gene, modifications which can be used for a diagnostic approach. Briefly, the protein may be truncated, reduced in size or absent; its properties, in particular its antigenicity, may be modified. All these modifications may be used in a diagnostic approach using several well known methods based on the use of mono- or polyclonal antibodies which recognize the normal protein or mutated variants, and this using the study of protein extracts or of tissue sections (for example skin biopsies), or studies carried out in vivo (imaging with the aid of antibodies coupled to a molecule which is detectable in PET-scan type imaging, and the like).

The polyclonal or monoclonal antibodies may be obtained by immunological reaction of a human or animal organism with an immunogenic agent consisting of a protein or a polypeptide capable of being obtained from prokaryotic or eukaryotic cells transformed by a vector as described above. Preferably, the immunogenic agent consists of a specific polypeptide of the mutated form of the Notch protein whose sequence is chosen from the polypeptide sequences comprising at least one mutation chosen from the mutations corresponding to FIG. 3 or to Table C.

The present invention finally relates to therapeutic compositions containing, as active ingredient, a compound with a pro-Notch3 activity, especially as described above, as well as therapeutic compositions containing, as active ingredient, a compound with an anti-Notch3 activity.

Other characteristics and advantages of the present invention will appear on reading the examples below, with reference to the accompanying drawings in which:

FIG. 1 reproduces the cDNA sequence of human Notch3 as well as the corresponding protein sequence,

FIG. 2 represents the general structure of the product of the Notch3 gene as well as the mutations which were detected by aligning the human cDNA clones with mouse Notch3,

-   -   A—mouse Notch3 gene with its 34 EGF domains, 3 Notch/Lin12         repeats and 6 cdc10 repeats, as well as the transmembrane         domain,     -   B—at the bottom, 8 of the human cDNAs with the identifications         corresponding to FIG. 1,     -   at the top, the alignment of some genomic sequences with the         cDNA of at least 29 exons,     -   the origins of the various fragments appear at the bottom of         FIG. 4;     -   the various clones are available commercially or through         libraries;         -   the clones 261623; 153875; 149693 are available from the             IMAGE consortium;         -   the clone C-32b03 is available from GENEXPRESS (Généthon,             Evry, France);         -   the clones p28-20; CNA-20 are available from CLONTECH;

FIG. 3 schematically represents the situation and the nature of the mutations involved in CADASIL;

FIG. 4 represents a “Northern blot” analysis, the Northern blots containing 2 μg per line of human poly (A⁺) DNA, from left to right:

-   -   of various brain tissues,     -   of various adult organs,     -   of various fetal organs;         they are hybridized with p28-20, a Notch3 human 1.45 kb cDNA         probe; a 7.5 to 9.5 kb transcript is detected in all the         tissues, both adult and fetal, with the exception of the liver,         the transcript is weakly expressed in the brain tissue in the         middle and on the right;         on the contrary, on the left, not only are transcripts of all         the tissues observed, but also the presence of a transcript of         between 2.4 and 1.3 kb whose presence has never been mentioned         and which may be of a very high importance is observed.

EXAMPLE 1

Scheme for the Isolation and Analysis of the Notch3 Gene

Following the remarks and the analysis which were summarized at the beginning of the description as regards the location of the gene, murine cDNA probes were used to isolate the cDNA for the human Notch3 gene, and then genomic clones whose sequences could be aligned with it and with the murine cDNA sequences.

Additional information on the sequences were obtained from a cDNA fragment (884Na4) obtained by cDNA selection on YAC (884 gl) and from two genomic fragments (J431NH and J432NH) which were obtained by subcloning of BAC 13J4 NotI-HindIII fragments.

In the screening of the (dBest) data bank with all the sequences, it has been possible to identify additional clones (IMAGE clones, Genexpress).

The coding sequence of the human Notch3 gene, which is highly homologous to the corresponding murine gene, is represented in FIG. 1.

Table A schematically represents the structure of the gene, specifying the sequence and the position of the exon-intron junctions.

In this table, the first exon corresponds to a sequence whose 5′ end was not completely cloned, likewise for exon 33.

It should be noted that alternative cDNAs may exist which correspond to the known phenomenon of alternative splicing.

This sequence contains 34 EGF domains, 3 Notch/lin12 repeats, as well as 3 cdc10 ankyrin-like repeats. The human and murine proteins exhibit 90.5% identity on the sequence currently available. A 1.45 kb partial human cDNA probe containing the EGF-like domains reveals a ubiquitous transcript in the fetal tissues, as well as in the human adult tissues whose size of between 7.5 and 9.5 kb is similar to the murine transcript (FIG. 4).

This probe reveals another transcript in certain subregions of the brain whose size is estimated at between 1.3 and 2.4 kb (FIG. 4).

EXAMPLE 2

Study of the Mutations

In order to study the extent of the mutations in the Notch3 gene on CADASIL, the possible presence of a substantial genomic DNA rearrangement was first studied using various combinations of enzymes and of Notch3 probes.

No drastic rearrangement could be detected in the CADASIL patients, that is why point mutations were then tested for.

Thus, the mutations of the total coding sequence of the Notch3 gene in the genomic DNA were studied using a combination of SSCP method and heteroduplex analysis in 51 CADASIL patients with no family relationship. 28 of them belong to families fort which the evidence for a relationship with chromosome 19 has been demonstrated and 33 exhibit ultrastructural lesions of the wall of the arterioles of the skin (presence of osmiophilic granular deposits in the basal membrane of the vascular smooth muscle cell.

All the splicing junctions, except 3, were analyzed. In addition, direct sequencing of the PCR products of exon 4 and its splicing sites was carried out on all the patients.

Impairments which were compatible with corresponding mutations were found in 42 patients (82%), said mutations not being observed in any of the 200 control chromosomes. For 26 patients, it was possible to analyze one or more which were related, affected or otherwise, and in each case it was established that the mutation segregated with the CADASIL phenotype. There are 29 different mutations, of which 20 are described for the first time. They include 24 missense mutations which appear in 40 patients, which mutations should replace (16) one amino acid with an additional or mutated cysteine (8) one of the 6 cysteine residues, which are the key elements of the EGF domains.

Two of the mutations at the 5′ splicing site in the latter two patients should normally affect the splicing of exon 4. The last three mutations are missense mutations which appear in 3 patients simultaneously with the mutations described above.

Patient 21 carries 2 distinct mutations which change an arginine to cysteine at codon 141 in EGF 3 and which changes a conserved glycine to alanine at codon 288 in EGF 7. This patient's pedigree was not available; it was not therefore possible to study the cosegregation of these two mutations.

Patient 29 carries a first mutation in EGF 4 which changes an arginine 182 to cysteine and a second which changes a highly conserved alanine 1852 to threonine in the cdc10 domain. These two mutations segregated with the disease.

The last patient 55 is a carrier of two distinct mutations in the EGF domains, which change a cysteine (224) to a tryptophan and a nonconserved leucine (497) to a serine residue.

Although the latter three missense mutations are not detected in the 200 control chromosomes, they may involve rare polymorphisms given the presence also of missense mutations which mutate or create cysteine residues.

It should be noted that most of these 26 mutations having a pathogenic effect lie exclusively in the EGF parts. 41% of these mutations (11 out of 26) appearing in 25 patients are situated in exon 4 and 65% (17 out of 26) lie in the first 6 EGF domains (see in particular FIG. 3.

FIG. 3 allows the detection to characterize the main mutations detected, the nomenclature chosen indicates the position of the mutation as well as the corresponding modifications of the protein.

As was indicated above, the fact that a Notch gene is involved in neurological disorders in adults appears completely surprising since Notch is mainly known to be involved during development in drosophila. None of the CADASIL families studied up until now exhibits developmental abnormalities.

EXAMPLE 3

Detection of the Mutations in Patients by the SSCP Method

The oligonucleotides used as primers were synthesized from intron-exon joining sequences (Table B) so as to amplify genomic fragments of about 200 bp. The sequences of the PCR primers are given below (Table A).

The analyses can be carried out using DNA extracted from blood samples or any other tissue.

The amplification reactions are carried out in a final volume of 25 μl containing 100 ng of genomic DNA, 0.5 μm of each primer, −150 μg of a mixture of 4dNTPs, Taq polymerase 1×PCR from Cetus, 1 U Taq polymerase (BRL), 1.5 μCi αdCTP labeled with P33 according to a protocol comprising 30 identical cycles (94° C., 15 s; 650° C., 15 s; 72° C., 15 s).

For some of the primers, an “annealing” temperature of 70° C. should be used, as indicated in Table A.

The PCR products are denatured in 50% formamide and separated by electrophoresis in a 6% nondenaturing polyarylamide gel.

After autoradiography, the SSCP bands obtained in the patients are compared with those of healthy controls in search of abnormal variants. Their analysis can be used as a diagnostic approach. These variants can then be sequenced if necessary.

TABLE A Exon-intron structure of the Notch3 gene (sequences and positions of the exon-intron junctions) Splice Acceptor Site Exon Splice Donor Site Intron/Exon (Size) Position Exon/Intron 1     1–196 CTGCAC/gtgaggggc IaAlaAI cccacacag/CCCCCC 2(79)   197–275 CTGCCT/gtgagtgcc           aProPr aCysLc  gcccacag/GTGCCC 3(143)   276–418 TCCGAG/gtgagagg           uCysPr heArgG  ccctccag/GCCCTG 4(339)   419–757 TTCCTG/gtgagtga           lyProA euProG  cttgttag/GGTTTG 5(123)   758–880 GGACAG/gtgggcac           lyPheG rpThrG  tgccacag/GCCAGT 6(234)  881–1114 AGACTG/gtgagtgg           lyGlnP ysThrG  cttcccag/GCCTCC 7(156) 1115–1270 CTATCG/gtgagggg           lyLeuL erIleG  tccggcag/GCGCCA 8(187) 1271–1456 TGGCAG/gtgggtgg           lyAlaA etAlaG  tgccccag/GCTTCA 9(114) 1457–1570 CCTCGG/gtgaggac           lyPheT roSerG  caccccag/GCTTCA 10(114) 1571–1684 CCGAGG/gtgaggcg           lyPheS laGluG  ccccacag/GCTTTG 11(234) 1685–1918 CCACAG/gtgggacc           lyPheG hrThrG  gcccctag/GTGTGA 12(111) 1919-2029 TCACAG/gtgggcaa           lyValA heThrG  ctccccag/GGCCCC 13(193) 2030–2222 TGGCGG/gtgagggc           lyProL oGlyGl  cctgccag/GTTCCG 14(152) 2223–2374 TCCAGG/gtgtgtac           yPheAr alGlnG  cccaacag/GACGTC 15(114) 2375–2488 GGCAAG/gtatgccac           lyArgG rpGlnG tacccccag/GCCCAC 16(156) 2489–2644 ACCCCA/gtgagtgca           lyProA spProA  gtccgcag/ACCCAT 17(226) 2645–2870 CCCCAG/gtgggcgg           snProC rProSe  cgctccag/CTCCTG 18(202) 2871–3072 TGCCAG/gtgggtgg           rSerCy CysGln  ccctccag/ACGCTG 19(148) 3073–3220 AGATCG/gtgagtgg           ThrLeu InIleG  ctttgcag/GGGTGC 20(185) 3221–3405 TGTGAG/gtaagggg           lyValA CysGlu  cactgaag/TGTCTT 21(133) 3406–3538 CGCTGG/gtatgcca           CysLeu hrLeuG  tcccccag/GGGTGC 22(258) 3539–3796 TCTCAG/gttaacct           lyValL hcSerG  tcgctcag/GTCCTC 23(119) 3797–3915 GCCCAG/gtaggtgtg           lyProA AlaGln gacccccag/CCGTTC 24(566) 3916–4481 TTGCAA/gtgagccc           ProPhe rCysAs  cccaccag/CCCGGT 25(333) 4482–4814 GATCGG/gtgagtgac           nProVa lIleG tccctgcag/CTCGGT 26(155) 4815–4969 TGCGGG/gtgcggcc          ySerVal alArgG tgctcttag/GGGAGC 27(223) 4970–5192 CATGAA/gtgagaac           lyGluP yMetLy  tccgccag/GAACAT 28(85) 5193–5277 CTAAAG/gtactgcc           sAsnMe LeuLys cccctccag/GTAGAG 29(162) 5278–5440 GCCCAG/gtcagtgac           ValGlu lyProA  ccctgcag/ATGGCT 30(305) 5441–5745 TTCCAG/gtgagata           spGlyP PheGln  tgtcctag/ATTCTC 31(148) 5746–5893 AGCTTG/gtaggttg           IleLeu luLeuG  ccctccag/GGAAAT 32(99) 5894–5992 AGCAAG/gtgagccc           lyLysS SerLys  ccccccag/GAGGAG 33 5993–           GluGlu

TABLE B Sequences of the primers used for the screening of the mutations of the Notch3 gene Exon Size Domain Primers PCR product size 1 Signal EOF AAGGAGGGAGGAGGGGAG 125 peptide EOR TGGGGGTTCTTGCACTCC* EOF AAGGAGGGAGGAGGGGAG 163 E0RBIS GGTTCCTGCCTCCCATGA* 2 79 EGF1 E1F TCCTCCACCTTCCTTCAC* 148 E1R ACACACAGGGCCCACTGGT* 3 143 EGF 1-2 N1F TGTGCTGCCCAACCAAGCCA* 224 N1R ACTGACCACACCCCCGACTA* 4 339 EGF 2-5 N2AF TAGTCGGGGGTGTGGTCAGT* 192 N2AR TCATCCACGTCGCTTCGGCA CNAF ATGGACGCTTCCTCTGCTC 167 CNAR ACATAGTGGCCCTGTGTAGC CNAF ATGGACGCTTCCTCTGCTCC 295 N3AR CCTCTGACTCTCCTGAGTAG* 5 123 EGF 5-6 N23Fbis TGACCATCCTTGCCCCCTT* 241 N23R CTGGCCTGTGGCACACAGAT* 6 234 EGF 6-8 N13AF TGGACTGCTGCATCTGTGTG* 191 NI3AR ACACGCCTGTGGCACAGTCA N13BF GAGCTGCAGTCAGAATATCG 145 N13BR ATCCATGGCTCCCTGCAGAG* 7 156 EGF 8-10 N24F CAGAGCAGGAAGATCTGCCT* 229 N24R CATTCACAGACGACGGAGCT* 8 187 EGF 10-11 N3F ATCGCACTCCATCCGGCA* 212 N3R ACCCACCTGCCATACAGA* 9 114 EGF 11-12 N25AF CGTTCACACCATAGGGTAGC* 215 N25AR CCCCTTCCCAGACATGTCTT 10 114 EGF 12-13 N2SBF CTTGTCGGACTGTCATTGG 195 N25BR GTGTACTGCTCTCACCCTT* 11 234 EGF 13-15 N4AF ATTGGTCCGAGGCCTCACTT* 213 N4AR ACCTGGCTCTCGCAGCGTGT N4BR CCATTCCCAACCCCTCTGTG 199 N4BF TGCCTGTGCTCCTGGCTACA* 12 111 EGF 15-16 N5F TGGCCACTCCATGCCATGTT* 166 N5R TCTCATGGCAGCCACTTGCC* 13 193 EGF 16-18 N14F ATGAGTGTGCTTCCAGCCCA* 258 N14R GCAGTGTCTGAGGCTGAGAA* 14 152 EGF 18-19 N6F TCCCTGGCCTGACTACCTTC* 207 N6R CTGCAGAGGGAAGGTGAGGT* 15 114 EGF 19-20 N26BR AAGGCTATCCTGCTTCC* 183 N26BR GAGGAGGAGGGAAGAGAA* 16 156 EGF 20 22 S13FBIS AGGATGTGGACGAGTGTGCT 195 N26CR GCTTAATGACTGTGTTCC* 17 226 EGF 22-24 N15AR TCAGACTGGGCTAATGGGGG* 257 N15AR TCGCAGTGGAAGCCTCCGTA N15BF GATGTGGATGAGTGCCTGAG 166 N15BR GTCCTGCTCTTCAAGCAGA* 18 202 EGF 24-25 N27F GATCCTCCCTCCCACTCCTT* 256 N27R AGGTCCCCAGTAACTCCA* 19 148 EGF 25-27 N22F ACTGACTCTAAGTGCTTCCC* 240 N22R AGCAGGAGGTACGTGCATGA* 20 185 EGF 27-28 N7F TGTTCCTGTGCCACTCTCCT* 249 N7R ACCTCCTCTTCCCTCTCCT* 21 133 EGF 28-29 N8F TCTGTGTCCCACTAAGCTGA* 237 N8R CAAGAGGAAATGAAGACAGC* 22 258 EGF 29-31 N9AF TTCCTCTTGACCACCCCTCG* 217 N9AR TGGCAGGCACCTGAGCGACA N9BF CAGGATACACTGGTTTGCGC 209 N9BR TGCCACGTTATGGATCAGCC* 23 119 EGF31-32 N10F GATCTACATGCTCCCGCTCG* 178 N10R TACTCCTCCTCCATAGGCCG* 24 566 EGF 32-34 N16AFTR CGTTCTGGGGTCCGCGTT 249 Lin12 N1-3 N16DR AAGCGCAGCGGAAGAAGGGC N16FF GCCCTTCTTCCGCTGCGCTT 230 N16FR ACTGCAGCGCCTCGCATTGC N16GF CTGCGACCGCGAGTGCAACA 239 N16HR ATAGACAGACGGATCGAT* 25 331 Lin12 N3 N21CF CTCTCTGCCTCACCCTT* 207 N21CR GCTGGAACGCAGTAGCT N21DF TGCTCACAGTGCTGCTG 223 N21DR CACGGCTTTTCCAGGTG* 26 155 N34F TTTGAGCCCTCTGGTCC* 306 N34R AAGAGCAGGAAGCAGAG* 27 222 TM N28Fbis TCCCTCTGCTTCCTGCTCTT* 291 N28R TCACAAGGTCCCCGTAGTCA* 28 85 J5N3F CTCACATCCCCTCTTCCCAT* 203 J5N3R ATCACGCCCATCATCCACTG* 29 163 Cdc10 N1 L24bisf CAGCACCAAAGGGTG* 241 L24bisR CATCCCTTTGGGAGG* 30 305 Cdc10 N1-3 N17AF ATGGCTTCACCCCGCTAATG 176 N17AR AGCCAGGTGCAAAGCAGTCT N17BF TCAGCTTGGGGCACGGACTG 165 N17BR GCATCGGCTGTGACAGCTGT 31 148 Cdc10 N4-5 N26FBIS TCTTCCTGCCATGACCCCT* 239 N26RBIS CAGGTGACACTAACCCAGTC* 32 98 Cdc10 N5-6 N31F TCCTGACCTCTCTCCCCTTC* 178 N31R AATGGCGCTGTGCCACTGCT* 33 Cdc10 N6 N32AF GCTACTGTTAGCTGGGCTTT* 214 NLS N32AR TGATCCAGCAAGCGCACGAT PEST N32EFTER TCACCGACCACCTGGACA 425 N32DR ACCAAGCTGTGCCAGAGA N32DF TCCAAGAAGAGCAGGAGG 246 N32DR ACCAAGCTGTGCCAGAGA N32BF CAGTGTCTCTGGCACAGCTT 248 N32BR TCCTGGGACTGCCAGGTAA N32CF AGCTGCTCAACCCAGGGA 229 N32CR GTGGATTCGGACCAGTCT N32GF GAATCCCCTGAGCACT 235 N32GR CTAAGAACTGACGAGC *intronic primers

TABLE C Notch3 mutations in CADASIL patients Evidence of SMC Notch3 Patient linkage lesions nt^(a) Notch3 mutation Effect Exon Domain Segregation 52* nd nd 224 TGT-->TAT C₄₉-->Y* N2 EGF1 nd 56 nd + 291 TGG-->TGT W₇₁-->C N3 EGF1 nd 11 + nd 406 CGT-->TGT R₁₁₀-->C N3 EGF2 +  3 + + 419(−2) AG-->GG abnormal splicing N4 + of exon 4 ? 39 nd + 419(−2) AG-->CG abnormal splicing N4 nd of exon 4 ? 10 + + 475 CGC-->TGC R₁₃₃-->C N4 EGF3 + 20 nd + 475 CGC-->TGC R₁₃₃-->C N4 EGF3 nd 46 + nd 475 CGC-->TGC R₁₃₃-->C N4 EGF3 +  6 + nd 499 CGC-->TGC R₁₄₁-->C N4 EGF3 + 12 + + 499 CGC-->TGC R₁₄₁-->C N4 EGF3 + 19 + nd 499 CGC-->TGC R₁₄₁-->C N4 EGF3 + 21* nd + 499 CGC-->TGC R₁₄₁-->C N4 EGF3 nd 941 GGC-->GCG G₂₈₈-->A* N5 EGF7 nd 38 + nd 499 CGC-->TGC R₁₄₁-->C N4 EGF3 + 49 + + 499 CGC-->TGC R₁₄₁-->C N4 EGF3 + 26 + + 514 TGC-->CGC C₁₄₆-->R N4 EGF3 +  4 + + 535 CGC-->TGC R₁₅₃-->C N4 EGF3 + 50 nd + 535 CGC-->TGC R₁₅₃-->C N4 EGF3 +  9* + + 583 CGC-->TGC R₁₆₉-->C* N4 EGF4 + 15* + nd 583 CGC-->TGC R₁₆₉-->C* N4 EGF4- + 24 + nd 583 CGC-->TGC R₁₆₉-->C N4 EGF4 + 36* nd + 583 CGC-->TGC R₁₆₉-->C* N4 EGF4 nd 48 nd + 583 CGC-->TGC R₁₆₉-->C N4 EGF4 nd  1 + nd 589 GGT-->TGT G₁₇₁-->C N4 EGF4 + 45* + + 622 CGC-->TGC R₁₈₂-->C* N4 EGF4 + 47* nd + 622 CGC-->TGC R₁₈₂-->C* N4 EGF4 nd 29* + + 622 CGC-->TGC R₁₈₂-->C N4 EGF4 + 5632 GCT-->ACT A₁₈₅₂-->T* N30 cdc10 + 41 nd + 631 TGT-->CGT C₁₈₅-->R N4 EGF4 nd 57 nd + 712 TGC-->AGC C₂₁₂-->S N4 EGF5 nd  8 + nd 742 TGT-->GGT C₂₂₂-->G N4 EGF5 + 55 nd nd 749 TGT-->TAT C₂₂₄-->Y N4 EGF5 + 1568 TCG-->TTG S₄₉₇-->L N9 EGF12 − 14 + + 851 TAT-->TGT Y₂₅₈-->C N5 EGF6 + 54* nd + 1703 TGT-->TAT C₅₄₂-->Y* N11 EGF13 nd 17* + + 1750 CGC-->TGC R₅₅₈-->C* N11 EGF14 + 18* + + 1750 CGC-->TGC R₅₅₈-->C* N11 EGF14 + 31* nd + 1810 CGC-->TGC R₅₇₈-->C* N11 EGF14 + 43 nd nd 2260 CGC-->TGC R₇₂₈-->C N14 EGF18 nd 25 + + 3031 CGC-->TGC R₉₈₅-->C N18 EGF25 + 42 nd + 3031 CGC-->TGC R₉₈₅-->C N18 EGF25 nd  7 + nd 3094 CGC-->TGC R₁₀₀₆-->C N19 EGF26 + 35 nd nd 3169 CGC-->TGC R₁₀₃₁-->C N19 EGF26 + 33 nd nd 3769 CGC-->TGT R₁₂₃₁-->C N22 EGP31 nd 58* nd + 3859 TGC-->CGC C₁₂₆₁-->R* N23 EGF32 nd *patient and mutation previously reported⁷ SMC: smooth muscle cell

REFERENCES FOR THE VARIOUS METHODS CITED ABOVE

Polymerase Chain Reaction (PCR)

-   Saiki et al., Science 239, p. 487, 1988+reference manual.     SSCP -   Orita et al., Proc. Natl. Acad. Sci. USA, 86, p. 2766–2770, 1989.     Techniques for Detection of Mutations Based on the Demonstration of     Mismatches     -   chemical cleavage     -   enzymatic cleavage (S1 nuclease)     -   heteroduplex     -   Allele Specific Oligonucleotide probes (ASO)

REFERENCES

-   Cotton et al., Proc. Natl. Acad. Sci. USA, 85, 4397, 1988 -   Sherk et al., Proc. Natl. Acad. Sci. USA, 72, 989, 1975 Cariello,     Hum. Genet., 42, 726, 1988     Cloning Vectors and Basic Molecular Biology Techniques -   Current protocols in molecular biology, Eds F. M. Ausubel, R.     Brent, R. E. Kingston, D. D. Moore, S. G. Seldman, J. A. Smith     and K. Struhl, Published by Green Publishing Associates and Wiley     Interscience, 1st edition 1987, John Wiley and sons -   Molecular cloning. A laboratory manual, J. Sambrook, E F Fritsch     and T. Mariatis, 2nd edition, 1989, Cold Spring Harbor Laboratory     Press 

1. An isolated or purified nucleic acid encoding, or complementary to a nucleic acid encoding, a polypeptide comprising an amino acid sequence that only differs from SEQ ID NO:2 by one or more mutation selected from the group consisting of R133C, R141C, C146R, R153C, R169C, R182C, Y258C, R558C, and R985C.
 2. The isolated or purified nucleic acid of claim 1, wherein the encoded polypeptide contains the mutation R141C in SEQ ID NO:
 2. 3. A nucleic acid primer comprising a nucleic acid sequence that complements the purified nucleic acid sequence of claim 1 at a region that encodes a mutation or mutations selected from the one or more mutation.
 4. A nucleic acid probe that hybridizes under stringent conditions to the purified nucleic acid sequence of claim 1 at a region that encodes a mutation or mutations selected from the one or more mutation, wherein said stringent conditions comprise a hybridization step at 68° C. in 6×SSC buffer, 5× Denhardt's solution, 0.5% SDS, and 100 μg/ml of salmon sperm DNA, a 5 minute washing step at room temperature in 2×SSC and 0.1% SDS, a 15 minute washing step at 37° C. in 0.1×SSC and 0.5% SDS, and a 30 minute washing step at 68° C. in 0.1×SSC and 0.5% SDS buffer.
 5. A reagent for detecting or identifying a nucleic acid that encodes a polypeptide that only differs from SEQ ID NO: 2 by one or more mutation selected from the group consisting of R133C, R141C, C146R, R153C, R169C, R182C, Y258C, R558C, and R985C, wherein said reagent comprises the probe according to claim
 4. 